Extended field iterative reconstruction technique (efirt) for correlated noise removal

ABSTRACT

Computerized method and system for improving 3D reconstruction images involves applying the Extended Field Iterative Reconstruction Technique (EFIRT) to remove correlated noise, in addition to using COMET (constrained maximum relative entropy tomography) to eliminate uncorrelated noise, wherein the EFIRT is applied by performing a set of successive reconstructions on an extended field larger than a region of interest (ROI); and extracting and averaging the ROI from said set of successive reconstructions.

TECHNICAL FIELD

The present invention relates to a computer implemented method andsystem for removing correlated noise by applying Extended FieldIterative Reconstruction Technique (EFIRT) in three-dimensional (3D)structure reconstructions.

BACKGROUND ART

Nowadays tomographic reconstruction has become a widely used techniquefor structure determinations in medical and biological applications.Macromolecules such as proteins and nucleic acids are importantbiological macromolecules, which possess important functionalinformation within their structure. In addition, most biologicalmacromolecules including proteins are flexible, with enhanced ability tointeract with other molecules. As such, structure determination hasimportant applications especially for purposes of understandinginteractions between molecules, and thus, is of vital importance in drugdevelopment.

Many standard 3D structure determination techniques, such as x-raycrystallography, nuclear magnetic resonance (NMR) or single particlecryo-electron microscopyare, are based on averaging, which may causelosses of most information about the conformation or flexibility of themacromolecular structure. To overcome this problem, cryo-electrontomography (Cryo-ET) is introduced, which involves taking transmissionelectron microscope (TEM) images of a cryo-specimen at different tiltangles and reconstructing a 3D tomogram through aligning and backprojecting the different images. Cryo-ET preserves the native structureof molecules due to rapid freezing to liquid nitrogen temperature, anddoes not necessarily involve averaging. As a result, it enables athorough study of flexible multi-domain proteins in the native state.

Cryo-ET 3D reconstructions of macromolecules encounter several types ofnoise. One type of noise is specimen noise, which is mainly due torearrangement of the specimen during data recording or degradation dueto electron beam damage. This type of noise is normally minimized usinga sufficiently low dose. The low dose, however, increases theuncorrelated shot noise caused by low illumination. In addition,correlated noise can appear due to imperfections of the TEM detector.This is because, normally, a gain reference is created to equalize theresponses from individual detector elements, but errors in the gainreference can give rise to noise that is correlated with a region of thedetector rather than with the specimen.

Most uncorrelated shot noise in cryo-ET reconstructions can besignificantly reduced using procedures for regularized refinement, suchas constrained maximum entropy tomography (COMET). In medicalapplications the shot noise is generally avoided to a large extent sincea high dose could be used. However, correlated noise emanating from thedetector measurement is difficult to handle since this kind of noise isrelated to sensitivity variations across the detector surface. To ensurehomogenous signal responses, flat-fielding is often used in practice,but given the variations in the quality of flat-fielding, the resultingbase-line comes with variations. The variations around the base-lineresult in a position-correlated noise, which, in a 2D or 3Dreconstruction, causes an increased background in a position dependenton the length of the direction orthogonal to the detector surface, orfor many projections, in the untilted beam direction.

In light of the above, there is a need for improved techniques to removecorrelated noise for enhanced three-dimensional (3D) reconstructionimages.

SUMMARY OF INVENTION Technical Problem

Accordingly, the present invention is directed to a scheme thatsubstantially obviates one or more of the above-discussed and otherproblems due to limitations and disadvantages of the related art.

An object of the present invention is to provide a computer implementedsystem and method for removing correlated noise in 3D reconstructions.

Solution to Problem

To achieve these and other advantages and in accordance with the purposeof the present invention, as embodied and broadly described, in oneaspect, the present invention provides a computer implemented method forremoving correlated noise by applying Extended Field IterativeReconstruction Technique (EFIRT) in a three-dimensional (3D)reconstruction, wherein the method comprises performing a set ofsuccessive reconstructions on an extended field larger than a region ofinterest (ROI); and extracting and averaging the ROI from said set ofsuccessive reconstructions.

In one respect, the present invention provides a computer implementedmethod including the above-referenced features, wherein the set ofreconstructions is performed by increasing volumes of each successivereconstruction by increasing the value of z in the direction of theelectron field.

In another respect, the present invention provides a computerimplemented method including the above-referenced features, and furthercomprising removing uncorrelated noise in said 3D reconstruction withCOMET (constrained maximum relative entropy tomography).

In yet another respect, the present invention provides a computerimplemented method including the above-referenced features, wherein the3D reconstruction is implemented via a Cryo-Electron Tomography(Cryo-ET) application.

In one respect, the present invention also provides an apparatus forimproving a three-dimensional (3D) reconstruction of a sample, saidapparatus programmed to execute instructions including: removinguncorrelated noise in said 3D reconstruction with COMET (constrainedmaximum relative entropy tomography); and removing correlated noise insaid 3D reconstruction by applying an Extended Field IterativeReconstruction Technique (EFIRT) procedure.

In another respect, the present invention provides an apparatusincluding the above-referenced features, wherein said EFIRT procedurecomprises performing a set of successive reconstructions on an extendedfield larger than a region of interest (ROI); and extracting andaveraging the ROI from said set of successive reconstructions.

Advantageous Effects of Invention

Additional or separate features and advantages of the invention will beset forth in the descriptions that follow and in part will be apparentfrom the description, or may be learned by practice of the invention.The objectives and other advantages of the invention will be realizedand attained by the structure particularly pointed out in the writtendescription and claims thereof as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory, andare intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating how to apply Extended Field IterativeReconstruction Technique (EFIRT) in structure reconstructions accordingto an embodiment of the present invention.

FIG. 2 is a diagram providing an alternative illustration how to applyEFIRT in structure reconstructions according to an embodiment of thepresent invention.

FIG. 3 illustrates an example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 4 illustrates an example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 5 illustrates an example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 6 illustrates an example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 7 illustrates an example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 8 illustrates another example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 9 illustrates another example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 10 illustrates another example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention.

FIG. 11 illustrates an algorithm for improving 3D reconstructionperformance by removing uncorrelated and correlated noises according toan embodiment of the present invention.

FIG. 12 is a diagram illustrating a computer or data processor forimplementing the algorithm of FIG. 11 according to an embodiment of thepresent invention.

DESCRIPTION OF EMBODIMENTS

The present invention improves data processing in structurereconstructions, and more specifically, in some embodiments, provides acomputer implemented method and system for removing correlated noise dueto detector imperfection in 3D reconstructions.

In some embodiments of the present invention, the primary aspects of thesystem are the following.

EXAMPLE 1

An algorithm for improving 3D reconstruction images comprises firstremoving the shot noise or uncorrelated noise with COMET (constrainedmaximum relative entropy tomography), and furthermore, eliminating theresidual noise, including correlated noise from detectors, by applyingthe EFIRT procedure. Notably, such algorithm can be implemented in dataprocessing software, or a data processor or computer installed with saiddata processing software. Additionally, such algorithm can be applied invarious applications including, without limitation to, Cryo-ElectronTomography (Cryo-ET) and other 3D reconstruction procedures/apparatus.It should be noted that in some embodiments, the removal of shot noiseor uncorrelated noise prior to applying EFRIT to remove correlated noisecan be accomplished in various ways, including, without limitation to,COMET. In other embodiments, the given data set may not containsignificant shot noise, and so it is unnecessary to remove the shotnoise before executing EFRIT to remove correlated noise. In other words,executing EFRIT to remove correlated noise can be a procedureindependent of the removal of uncorrelated noise.

EXAMPLE 2

An algorithm for removing correlated noise from detectors by EFIRTcomprises performing a set of reconstructions on an extended fieldlarger than the region of interest (ROI), followed by extracting andaveraging the ROI from the set of reconstructions. In one embodiment, inperforming the set of reconstructions, the volume of each successivereconstruction is increased by increasing z (direction of the electronfield). During extraction and averaging of the ROI, most correlatednoise is either cut out or canceled.

Usually shot noise in Cryo-ET reconstructions can be significantlyreduced using procedures for regularized refinement, such as constrainedmaximum entropy tomography (COMET), which essentially is an iterativealgorithm for enhancing the contrast and resolution in cryo-ET byincreasing the SNR. COMET can improve the fidelity of 3D reconstructionsby filtering out most of the noise. Briefly speaking, COMET works in thefollowing three steps:

Step 1

The density of the 3D reconstruction is modified in the iterations toincrease (theoretically maximize) the entropy of the existingreconstruction. If the reconstructed normalized density is given by

F({right arrow over (X)})

and

m({right arrow over (X)})

is the prior or estimated probability distribution of the density, thenthe entropy function to be maximized can be given by the followingequation:

${S(F)} = {- {\int{{F\left( \overset{\rightarrow}{X} \right)}{\ln \left( \frac{F\left( \overset{\rightarrow}{X} \right)}{m\left( \overset{\rightarrow}{X} \right)} \right)}{^{3}\overset{\rightarrow}{X}}}}}$

Step 2

Virtual projections are calculated from the reconstruction (i.e. thereconstruction from the previous iteration) in the direction of thetilts and a “goodness-of-fit” measure is determined. A good way todetermine this measure is by using the statistical reduced chi-squarefunction. As a general rule, a good fit is achieved with a reducedchi-square value of 1.0. Assuming that

F_(calc) ^((t))({right arrow over (X)}^((i)))

is the calculated density for the i^(th) view/tilt and

F_(obs) ^((t))({right arrow over (X)}^((i)))

is the same for the observed, then the chi square function can be givenby the following equation:

$\chi_{(i)}^{2} = {\frac{1}{N}{\sum\limits_{\overset{\rightarrow}{X}{(i)}}{\frac{1}{\sigma_{(i)}^{2}\left( {\overset{\rightarrow}{X}}^{(i)} \right)}\left\lbrack {{b^{(i)}*{F_{calc}^{(i)}\left( {\overset{\rightarrow}{X}}^{(i)} \right)}} - {F_{obs}^{(i)}\left( {\overset{\rightarrow}{X}}^{(i)} \right)}} \right\rbrack}^{2}}}$

The goal is to iteratively reduce the chi-square fitting parameter

X_((i)) ²

so that it can be as close as possible to 1.0.

Step 3

An optimal balance between the maximization of entropy and minimizationof chi-square function is determined and used to update thereconstruction before the next iteration.

COMET removes most of the shot noise from the reconstructions butcorrelated noise (usually from the detector) is still present. EFIRT isa technique to remove correlated noise by performing severalreconstructions on an extended field, which is generally larger than theregion of interest (ROI), and then averaging out the ROI from thesereconstructions. The extended field basically provides a “rubbish” binfor most of the noise as extending the field dilutes the noise in aspecific reconstruction. The extra region of the reconstruction outsidethe ROI enables the noise to spread out over a larger region, whichleads to less and less noise in the ROI. By averaging, the dilutioneffect on true signals within the ROI is compensated while the noise isreduced.

The execution of EFIRT in structure reconstructions can be explainedwith reference to FIG. 1. Consider the z-axis in FIG. 1 to be thevertical axis, i.e., the direction of the electron field, and let Z_(o)be the size of z for which the 3D reconstruction is desired.Reconstruction is performed for several increasing values of z such thatZ₁<Z₂<Z₃< . . . <Z_(n) resulting in ‘n’ number of 3D reconstructions. Asshown in FIG. 1, each successive reconstruction has an increased volumein the direction of z.

The ROI with z=Z_(o), which is the common region in all thereconstructions, is extracted from each reconstruction and averaged togive the effective reconstruction. Assuming that a single 3Dreconstruction with z=Z_(n) is represented by R_(z(n)) and the samereconstruction after extracting the ROI with z=Z_(o) is represented byR_(zo(n)), then EFIRT can be represented by the following equation.

$R_{eff} = {\frac{R_{{zo}{(0)}} + R_{{zo}{(1)}} + R_{{zo}{(2)}} + \ldots + R_{{zo}{({N - 1})}}}{N} = \frac{\sum\limits_{k = 0}^{N - 1}R_{{zo}{(k)}}}{N}}$

FIG. 2 demonstrates a 3D reconstruction from several 2D projections,which provides an alternative explanation of using the EFIRT procedurein removing correlated noise in 3D reconstructions. As seen in FIG. 2,the components of the 3D structure need to be present in all 2Dprojections so that it can be limited enough to form a rigid structure.However, noise does not have a representation in all 2D projections, andas a result, an unlimited structure can be formed, as shown in FIG. 2.When the same reconstruction is done on an extended field by increasingz, the rigid structure would remain, while the correlated noise would bedistributed over a larger volume and thus would have decreased presencein the ROI.

The net mass inside the ROI is both from the density of the signal andfrom that of the noise. As shown in FIG. 2, Ms represents the mass ofsignal and Mn is the mass of noise. Assuming that

P_(noise(1))

is the noise associated density with the first reconstruction, N is thetotal number of reconstructions and k is the factor by which the volumeis increased in each reconstruction by increasing z, then the net noisedensity of the final averaged reconstruction

ρ_(net noise)

is given by the following equation:

$\rho_{{net}\mspace{14mu} {noise}} = {\frac{\rho_{{noise}{(1)}}}{N} + {\frac{\rho_{{noise}{(1)}}}{kN}\left( {1 + \frac{1}{2} + \frac{1}{3} + \ldots + \frac{1}{\left( {N - 1} \right)}} \right)}}$

Based on this equation, the first term will decrease because N is in thedenominator. The second term has a harmonic series, which is a divergentseries, but it also has two terms in the denominator k and N, theproduct of which will always be larger than the harmonic series, therebydecreasing the overall factor. As such, the overall density of noisewill decrease by using EFIRT in 3D reconstructions.

FIGS. 3-7 illustrate an example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention. In this example,EFIRT is applied to the reconstruction of a protein receptor specimen,the glucocorticoid receptor (GR), from a low-dose Cryo-ET tilt series.Ten reconstructions were performed with the dimensions 250×250×250 to250×250×700 with z having an incremental increase of 50 grid points. Thecommon region 250×250×250 was cut out from all reconstructions and theaverage was calculated.

FIG. 3 shows volume renderings of the initial and final averaged EFIRTreconstructions. As seen in FIG. 3, the 3D reconstruction of this GRsample with EFIRT presents a higher image quality with less noise, ascompared to the 3D reconstruction without EFIRT.

To further visualize the effect of EFIRT, FIG. 4 shows thin slices ofboth the original and EFIRT averaged reconstructions and the differencebetween them. Clearly, the EFIRT-applied and averaged reconstruction isless noisy and has an enforced signal with higher SNR, as compared tothe original reconstruction 401. The difference 403 shows the amount ofnoise being removed.

The effect of EFIRT is further illustrated in FIG. 5, which presents thehistograms of the original and EFIRT averaged reconstructions and theircomparison and difference. As compared to the original histogram, it isclear that the averaged histogram has lower values, thus having a shifttowards the left and a higher peak. In the averaged and originalhistograms, the straight line of values in the beginning is a result ofCOMET, which converts all negative values to positive. Again, thedifference histogram shows the amount of noise being removed.

In executing the EFIRT procedure for 3D reconstructions, as the value ofz increases, the volume of each successive reconstruction alsoincreases, and the ROI has decreasing information content. As a result,the root mean square (RMS) deviation and mean value of the averagedreconstruction is supposed to be less for each successivereconstruction. This is demonstrated in FIG. 6 showing two graphs: onegraph shows the value of the RMS decreasing with respect to the value ofz, and the other shows mean value decreasing with respect to the size ofz.

FIG. 7 shows the cumulative density functions (CDF) of the original andthe averaged reconstructions. As seen in FIG. 7, the CDF of the averagedreconstruction, i.e., the first curve, goes up faster than the CDF ofthe original reconstruction, i.e., the second curve. This also showsthat the averaged reconstruction has reduced noise.

FIGS. 8-10 illustrate another example using EFIRT for 3D reconstructionsaccording to an embodiment of the present invention. In this example,EFIRT is tested for a non-biological sample, which usually does notexperience degradation due to electron exposure during imaging. Eightreconstructions were performed starting with 355×355×255 to 355×355×605with an increment in z of 50 grid points, and on electron micrographs ofSiO₂ nano particles. The common region 355×355×255 was cut out andaveraged from all reconstructions.

FIG. 8 shows the 3D reconstructions of the non-biological sample beforeand after applying EFIRT. As seen in FIG. 8, the EFIRT-appliedreconstruction presents a higher quality than the originalreconstruction. The effect of EFIRT is even more obvious in this examplebecause it is not degraded quickly during the TEM imaging, unlike theprevious example using a biological sample.

The effect of EFIRT is further visualized in FIG. 9, where thin slicesof the 3D reconstructions of a single SiO₂ nano particle are shown.Compared to the original reconstruction 901, the EFIRT-applied andaveraged reconstruction shows more information and a higher SNR.

Furthermore, FIG. 10 shows the histograms of the original reconstructionand the averaged reconstruction and their comparison and difference. Asshown in the comparison histogram, it is clear that the averagedhistogram has lower values than the original histogram, thus having ashift towards the left and a higher peak. Again, the differencehistogram shows the amount of noise being removed.

FIG. 11 illustrates an algorithm for improved 3D reconstruction imagesby removing uncorrelated and correlated noises according to anembodiment of the present invention. In brief terms, the algorithm inFIG. 11 comprises two steps: Step 1101, removing shot noise oruncorrelated noise with COMET (constrained maximum relative entropytomography); and Step 1102, removing correlated noise by applying theEFIRT procedure in 3D reconstructions. In one embodiment, as describedabove, in Step 1102, the EFIRT is applied by performing a set ofreconstructions on an extended field larger than the region of interest(ROI), followed by extracting and averaging the ROI from the set ofreconstructions. As the set of reconstructions is performed, the volumeof each successive reconstruction is increased by increasing z(direction of the electron field).

It should be noted that in some embodiments, the removal of shot noiseor uncorrelated noise prior to applying EFRIT to remove correlated noisecan be accomplished in various ways, including, without limitation to,COMET. In other embodiments, if the given data set does not containsignificant shot noise, then it won't be necessary to remove the shotnoise before executing EFRIT to remove correlated noise. In other words,executing EFRIT to remove correlated noise can be a procedureindependent of the removal of uncorrelated noise. Also, it should beunderstood that such algorithm can be applied in various applicationsincluding, without limitation to, Cryo-Electron Tomography (Cryo-ET) andother 3D reconstruction procedures/apparatus.

Notably, the algorithm in FIG. 11 can be implemented in data processingsoftware, or a data processor or computer installed with said dataprocessing software. FIG. 12 is a simplified diagram illustrating acomputer or data processor for implementing the algorithm of FIG. 11according to an embodiment of the present invention. It should be notedthat the computer 1200 is for illustration only, and many computercomponents included therein may not be shown or described in thefollowing paragraphs. As shown in FIG. 12, the computer 1200 comprises amemory 1210, a processor 1220 capable of accessing the memory 1210, andone or more I/O interfaces or other peripheral interfaces 1230 coupledto the processor 1220. Exemplary external or peripheral devices include,without limitation, a display 1240, a keyboard 1260, a camera 1280, aprinter or scanner in a combined or separate form 1250, a storage device1270 such as a USB or disk, and a microphone or speaker 1290. The memory1210 includes software programs or drivers for activating andcommunicating with each peripheral device. In one configuration, thesecomponents are connected through one or more communication buses (notshown) in the computer, which may include circuitry that interconnectsand controls communications between different components.

The memory 1210 includes high-speed random access memory, such as DRAM,SRAM, DDR RAM and/or other random access solid state memory devices; andincludes non-volatile memory, such as flash memory devices, a magneticdisk storage device, and/or other non-volatile solid state storagedevices. The memory 1210, or alternately non-volatile memory device(s)within the memory 1210, includes a non-transitory computer-readablestorage medium. While the memory 1210 is shown as being separate fromthe processor 1220, all or a portion of the memory 1210 may be embeddedin the processor 1220. In some embodiments, the memory 1210 stores thefollowing programs, modules and data structures, or a subset thereof: anoperating system 1212 that includes procedures for handling variousbasic system services and for performing hardware dependent tasks, andapplications 1214, including 3D reconstruction application 1214 a andcorresponding APIs 1214 b for processing data received from otherdevices and data to be transmitted to the other devices, securityapplications 1214 c, and any other applications. In some embodiments,the non-transitory computer-readable storage medium of the memory 1210includes instructions for performing all or a portion of the operationsas described herein. The processor 1220 is configured to access andexecute the instructions, programs, applications, and modules stored inthe memory 1210.

It will be apparent to those skilled in the art that variousmodification and variations can be made in the present invention withoutdeparting from the spirit or scope of the invention. Thus, it isintended that the present invention cover modifications and variationsthat come within the scope of the appended claims and their equivalents.In particular, it is explicitly contemplated that any part or whole ofany two or more of the embodiments and their modifications describedabove can be combined and regarded within the scope of the presentinvention.

REFERENCE SIGNS LIST

1200 Computer or Data Processor

1210 Memory

1220 Processor

1230 I/O interfaces or other peripheral interfaces

1240 Display

1250 Printer/scanner

1260 Keyboard

1270 Storage device

1280 Camera

1290 Microphone/speaker

1. A computer implemented method for removing correlated noise byapplying Extended Field Iterative Reconstruction Technique (EFIRT) in athree-dimensional (3D) reconstruction, said method comprising:performing a set of successive reconstructions on an extended fieldlarger than a region of interest (ROI); and extracting and averaging theROI from said set of successive reconstructions.
 2. The computerimplemented method according to claim 1, wherein the set ofreconstructions is performed by increasing volumes of each successivereconstruction.
 3. The computer implemented method according to claim 2,wherein the volume of each successive reconstruction is increased byincreasing the value of z in the direction of the electron field.
 4. Thecomputer implemented method according to claim 1, further comprisingremoving uncorrelated noise in said 3D reconstruction with COMET(constrained maximum relative entropy tomography).
 5. The computerimplemented method according to claim 1, wherein the 3D reconstructionis implemented via a Cryo-Electron Tomography (Cryo-ET) application. 6.The computer implemented method according to claim 1, wherein the 3Dreconstruction is applied to a biological sample or a non-biologicalsample.
 7. A system for removing correlated noise in a three-dimensional(3D) reconstruction by applying Extended Field Iterative ReconstructionTechnique (EFIRT), said system comprising: a processor; and a memoryaccessible to said processor, wherein said processor is programmed toexecute executable instructions stored in said memory, said executableinstructions, while executed, causing said processor to: perform a setof successive reconstructions on an extended field larger than a regionof interest (ROI); and extract and average the ROI from said set ofsuccessive reconstructions.
 8. The system according to claim 7, whereinsaid executable instructions, while executed, further causing saidprocessor to perform the set of reconstructions by increasing volumes ofeach successive reconstruction.
 9. The system according to claim 8,wherein the volume of each successive reconstruction is increased byincreasing the value of z in the direction of the electron field. 10.The system according to claim 7, wherein said executable instructions,while executed, further causing said processor to remove uncorrelatednoise in said 3D reconstruction with COMET (constrained maximum relativeentropy tomography).
 11. The system according to claim 7, wherein the 3Dreconstruction is implemented via a Cryo-Electron Tomography (Cryo-ET)application.
 12. The system according to claim 7, wherein the 3Dreconstruction is applied to a biological sample or a non-biologicalsample.
 13. An apparatus for improving a three-dimensional (3D)reconstruction of a sample, said apparatus programmed to executeinstructions including: removing uncorrelated noise in said 3Dreconstruction with COMET (constrained maximum relative entropytomography); and removing correlated noise in said 3D reconstruction byapplying an Extended Field Iterative Reconstruction Technique (EFIRT)procedure.
 14. The apparatus according to claim 13, said EFIRT procedurecomprising: performing a set of successive reconstructions on anextended field larger than a region of interest (ROI); and extractingand averaging the ROI from said set of successive reconstructions. 15.The apparatus according to claim 14, wherein the set of reconstructionsis performed by increasing volumes of each successive reconstruction.16. The apparatus according to claim 15, wherein the volume of eachsuccessive reconstruction is increased by increasing the value of z inthe direction of the electron field.
 17. The apparatus according toclaim 13, wherein the 3D reconstruction is implemented via aCryo-Electron Tomography (Cryo-ET) application.
 18. The apparatusaccording to claim 13, wherein the sample comprises a biological sampleand a non-biological sample.
 19. The apparatus according to claim 13,comprising a computer or data processor programmed with executableinstructions for data processing.
 20. The apparatus according to claim19, further comprising software programmed for performing structurereconstructions of different samples.